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refolding proteins using dialysis


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#1 chn09

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Posted 26 August 2010 - 09:08 PM

Hello everyone.
Im trying to purify alkaline phosphatase enzyme that has been overexpressed in E. coli BL21DE3. The protein formed inclusion bodies upon IPTG induction. I was able to isolate and purify the protein in the inclusion bodies using denaturing Nickel affinity chromatography. Then i used dialysis to remove the denaturant (8 M urea)

1. After i dialysed the unfolded protein (against 10 mM phospahte buffer containing 10 % glycerol and 1 mM 2-mercaptoethanol)for 48 hrs in a cold room. next day i was able to see a white precipitate at the bottom of the dialysis bag. I assumed it is the salt, but my colleague told it was the refolded protein itself.
2. I performed the assay to check the activity of refolded protein after concentrating the dialysed samples using a speed vac. Still that white precipitate was visible. And the assay worked.

Was that white precipitate a salt or refolded protein?. Is dialysis not enough to remove urea?
pls help

#2 protolder

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Posted 26 August 2010 - 09:49 PM

Hola, The denatured protein precipitate when you eliminate Urea, this is a commun phenomenon if the protein concentration is too high or/and the buffer donīt have the adecuates conditions of salt pH etc or the precipitated protein is the wrong structure folded protein. I suppose that this dialysis buffer is similar to the assay buffer.Moreover your assay runs but could be that the response was of the part of correct folded protein. So, if I were you I would try relationing the response with the protein concentration.
As refolding is a complication and your strain isnīt effectve to repress the expression, I would analize the soluble protein withouth induction , or would try induce at low inducer concentration because if there is any expression and it has a tag you have a better yield in quality without the step of refolding.Good luck




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